CN102696580A - Ultralow-temperature storage and regenerative-culture method for embedding and drying of isolated stem tips of test-tube horseradish plantlets - Google Patents
Ultralow-temperature storage and regenerative-culture method for embedding and drying of isolated stem tips of test-tube horseradish plantlets Download PDFInfo
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Abstract
The invention discloses an ultralow-temperature storage and regenerative-culture method for embedding and drying of isolated stem tips of test-tube horseradish plantlets, and relates to the technical field of germplasm preservation. The ultralow-temperature storage and regenerative-culture method comprises the following steps of: propagating materials, stripping the stem tips, preculturing, embedding calcium alginate into balls, drying the balls with sterile air flow, putting the dried balls into a freezing pipe, putting the freezing pipe in liquid nitrogen for ultralow-temperature storage, thawing, and restoring the regenerative culture. After ultralow-temperature storage, the isolated stem tips of the test-tube horseradish plantlets are treated by thawing, and the seedlings are directly formed after the regenerative culture is restored, so that the hereditary variation is avoided. The ultralow-temperature storage and regenerative-culture method is long-term, safe, stable, reliable and simple, and also has the advantages that the germplasm resource of the horseradish is effectively stored, the restoration and the recovery growth condition of the regeneration seedlings is favorable and the plant regeneration rate is high.
Description
Technical field
The present invention relates to germplasm preservation technology field, the particularly cryopreservation method of plant germplasm resource and cultural method again.
Background technology
In germ plasm resource preservation work,, still have higher regeneration rate when requiring the germplasm materials of long-term germplasm storehouse preservation after the several years, to breed again, and keep original genetic character in order to store the safety of germplasm.
Horseradish (
Eutrema wasabi(Sieboid) Maxim) have another name called scurvy grass; Be the herbaceos perennial that the Cruciferae scurvy grass belongs to, originate in East Asia, be distributed in provinces such as China southwest and Zhejiang; Be hold concurrently vegetables and medicinal important economic crops, its root, stem, leaf all have high economic worth.The horseradish complete stool has perfume (or spice), suffering, local flavor and sterilization idiocratic such as sweet, sticking, and its unique pungent taste makes it to be used for food as the flavouring of uniqueness, and it is edible that blade can be used as vegetables, effect such as sweating, diuresis are arranged, detoxify, purify the blood.Horseradish can promote starchiness food digestion, stablize vitamin C in the stomach and intestine, kill parasite in the digestive tract, research in recent years finds that also it has the effect of the cancer of the stomach of preventing and treating.Also have research report to claim that the horseradish crushed material adds that hydroperoxide can produce good biological phenol, have the effect of administering various chemical pollution things.
Horseradish is difficult for setting seeds, and its seed is to dry sensitivity, not low temperature resistant long term storage.Utilize offspring and tiller bud seedling to breed more in the production, need sand storing, overwintering, because the temperature and humidity conditions in the wayward storage; Perishable in the storage; Therefore horseradish germ plasm resource must be preserved in gene garden, field, carries out every year kind, receives, the storage operation, removes and takies great amount of time, human and material resources, financial resources and arable land; And be subject to the natural calamity influence, cause some kind disappearances.The test-tube plantlet method that use to exsomatize is preserved the horseradish germplasm needs continuous subculture, might be because of operational pollution, mix to make and preserve the material forfeiture, and prolong along with the holding time easily and morph.
Cryopreservation method is the best-of-breed technology means that long-term safety is preserved germ plasm resource, conserve space, and expense is low, and is stable, effective, worked out the method that multiple ultralow temperature is preserved germplasm at present.Because the different cultivars of different plant species even same species its suitable cryopreservation method and program technic is had nothing in common with each other; Currently also do not set up a kind of cryopreservation method with universality, this is one of ultralow temperature preservation technology main difficult point in practical application.Known cryopreservation method research about horseradish has two kinds of vitrifying method and embedding vitrifying methods, all has plant variation and the low problem of regeneration rate, the present research report of still not having more efficient methods.
Summary of the invention
Stem apex embedding seasoning ultralow temperature is preserved and cultural method again to the object of the present invention is to provide a kind of horseradish test-tube plantlet to exsomatize; Can be more safe and effective the germ plasm resource of preservation horseradish; And avoid producing genetic variation, the back shoot regeneration frequency that thaws is high, the regrowth well-grown.
To achieve these goals, technical scheme of the present invention is:
The stripped stem apex embedding seasoning ultralow temperature of horseradish test-tube plantlet is preserved and regeneration and cultivation method, may further comprise the steps:
-material expands numerous;
-stem apex strips;
-cultivate in advance;
-calcium alginate embedding balling-up;
The fluidized drying of-filtrated air;
-the cryovial of packing into;
-drop into nitrogen ultra low temperature to preserve;
-thaw;
-recovery regenerating and culturing.
Material expands numerous; Aseptic seedling is cultivated on the medium of MS+0.1mg/L BA+20g/L sucrose+7g/L agar of 825 mg/L NH4NO3,950mg/L KNO3,220mg/L CaCl22H2O, 185mg/L MgSO47H2O, 85mg/L KH2PO4; PH 5.8; 20 ± 2 ℃ of temperature, light application time 16h, per 35～40d subculture 1 time.
Stem apex strips, and the stripped stem apex that under aseptic condition, cuts the about 1～3mm of diameter from the aseptic seedling of robust growth is a material.
Cultivate in advance, the stem apex that strips is cultivated 1～2d with the MS solid culture medium that contains 0.3mol/L sucrose in advance under 20 ℃ or 25 ℃ of conditions.
Calcium alginate embedding balling-up; Stripped stem apex after cultivating is in advance placed the no calcium MS culture fluid that contains 25g/L sodium alginate+2mol/L glycerine+0.8mol/L sucrose; Draw the no calcium MS that contains a stem apex with aseptic dropper or pipettor and cultivate drop to the culture fluid that contains 0.1mol/L calcium chloride+2mol/L glycerine+0.8mol/L sucrose; And keeping 25～30 min to make it into the embedding ball in 25 ℃, the about 4～5mm of each embedding ball size contains a stem apex.
The filtrated air fluidized drying, the embedding ball that will contain stem apex takes out, and is positioned on the aseptic filter paper on the sterile petri dish dry 3～5h in filtrated air stream.
The cryovial of packing into, after the filtrated air fluidized drying, with the embedding ball cryovial of packing into, each cryovial is adorned 10～20 embedding balls.
The input nitrogen ultra low temperature is preserved, and the cryovial that the embedding ball is housed is placed drop into liquid nitrogen behind freezing pipe support or the sandbag and carry out ultralow temperature and preserve.
Thaw, from liquid nitrogen, take out cryovial, 1.5～3min thaws in 38～40 ℃ of water-baths.
Recover regenerating and culturing; Embedding ball after thawing is at MS+0.1mg/LBA+30g/L sucrose+6.5g/L agar of KNO3 950mg/L and NH4NO3 825mg/L; Recover in the medium of PH 5.8 to cultivate; After cultivating 8-10 days under the dark condition or the low light level, change over to again under the condition identical and cultivate earlier with successive transfer culture.
The invention provides a kind of safe, stable, reliably, simply, the method for long preservation horseradish germ plasm resource effectively; After the stripped stem apex of horseradish test-tube plantlet is preserved through ultralow temperature,, do not form callus with its processing of thawing, the direct Cheng Miao of recovery regenerating and culturing; Avoid producing genetic variation; Genetic stability is good, and regrowth recovers well-grown, and shoot regeneration frequency can reach more than 86%.
Description of drawings
Fig. 1 is the dry cryopreservation method step of the stripped stem apex embedding of horseradish test-tube plantlet block diagram.
Embodiment
Be further described below in conjunction with the Figure of description specific embodiments of the invention.
The stripped stem apex embedding seasoning ultralow temperature of horseradish test-tube plantlet as shown in Figure 1 is preserved and regeneration and cultivation method, comprises following concrete steps:
It is numerous that-material expands: aseptic seedling is cultivated on the medium of MS+0.1mg/L BA+20g/L sucrose+7g/L agar of 825 mg/L NH4NO3,950mg/L KNO3,220mg/L CaCl22H2O, 185mg/L MgSO47H2O, 85mg/L KH2PO4; PH 5.8; 20 ± 2 ℃ of temperature; Light application time 16h, per 35～40d subculture 1 time.Find in the test, the content of NH4NO3, KNO3, CaCl22H2O, MgSO47H2O, KH2PO4 in the MS medium is reduced by half, aseptic seedling growth and expand numerous respond well.
-stem apex strips: the stripped stem apex that under aseptic condition, cuts the about 1～3mm of diameter from the aseptic seedling of robust growth is a material.
-to cultivate in advance: the stem apex that strips is cultivated 1～2d with the MS solid culture medium that contains 0.3mol/L sucrose in advance under 20 ℃ or 25 ℃ of conditions.Make free water content reduction in the stem apex material cell, strengthen the freezing tolerance of stem apex material, prevent under the superfreeze condition, in cell, to form ice crystal.
-calcium alginate embedding balling-up: the stripped stem apex after will cultivating in advance places the no calcium MS culture fluid that contains 25g/L sodium alginate+2mol/L glycerine+0.8mol/L sucrose; Draw the no calcium MS that contains a stem apex with aseptic dropper or pipettor and cultivate drop to the culture fluid that contains 0.1mol/L calcium chloride+2mol/L glycerine+0.8mol/L sucrose; And keep 25～30 min to make it into the embedding ball in 25 ℃; About 4～the 5mm of each embedding ball size contains a stem apex.The drop that comprises a stem apex splashes in the culture fluid; The calcium ion reaction generates and solidifies the embedding ball in drop and the culture fluid; Further reduce the embedding ball and the water content of stem apex wherein than the culture fluid of hyperosmosis, prevent that further moisture generates ice crystal in the cell when superfreeze.
The fluidized drying of-filtrated air: the embedding ball that will contain stem apex takes out, and is positioned on the aseptic filter paper on the sterile petri dish dry 3～5h in filtrated air stream.On the basis of abovementioned steps, further reduce the moisture of embedding ball through filtrated air stream.
-the cryovial of packing into: after the filtrated air fluidized drying, with the embedding ball cryovial of packing into, each cryovial is adorned 10～20 embedding balls.
-dropping into nitrogen ultra low temperature to preserve: the cryovial that the embedding ball will be housed places and drops into liquid nitrogen behind freezing pipe support or the sandbag and carry out ultralow temperature and preserve.In ultralow temperature-196 ℃ liquid nitrogen, the activity of horseradish stem-tip tissue cell physiological stops, and can not produce genetic variation.
-thaw: from liquid nitrogen, take out cryovial, 1.5～3min thaws in 38～40 ℃ of water-baths.Thaw fast, prevent in the recovery process, to generate ice crystal stem apex material cell is damaged.
-recovering regenerating and culturing: the embedding ball after thawing is at MS+0.1mg/LBA+30g/L sucrose+6.5g/L agar of KNO3 950mg/L and NH4NO3 825mg/L; Recover in the medium of PH 5.8 to cultivate; After cultivating 8-10 days under the dark condition or the low light level, change over to again under the condition identical and cultivate earlier with successive transfer culture.Prove in the test; The embedding ball that will thaw places the MS medium of the potassium nitrate that reduces by half, ammonium nitrate content to regenerate, and the horseradish stem apex does not generate callus, has avoided the horseradish stem-tip tissue that the genetic variation phenomenon takes place when generating callus; It is stable that biological character keeps; Guarantee the safe and reliable preservation of horseradish germ plasm resource, recover the regrowth well-grown, shoot regeneration frequency can reach more than 86%.
To the biologic specificity of horseradish, this method cost is low, and operating performance is simply effective, and can be at lower cost long-term a large amount of preservation horseradish variety sources are practiced thrift soil and manpower and materials loss.
Claims (10)
1. the dry ultralow temperature of the stripped stem apex embedding of horseradish test-tube plantlet is preserved and regeneration and cultivation method, may further comprise the steps:
-material expands numerous;
-stem apex strips;
-cultivate in advance;
-calcium alginate embedding balling-up;
The fluidized drying of-filtrated air;
-the cryovial of packing into;
-drop into nitrogen ultra low temperature to preserve;
-thaw;
-recovery regenerating and culturing.
2. the dry ultralow temperature of the stripped stem apex embedding of horseradish test-tube plantlet according to claim 1 is preserved and regeneration and cultivation method; It is characterized in that: material expands numerous; On the medium of MS+0.1mg/L BA+20g/L sucrose+7g/L agar of 825 mg/L NH4NO3,950mg/L KNO3,220mg/L CaCl22H2O, 185mg/L MgSO47H2O, 85mg/L KH2PO4, PH 5.8,20 ± 2 ℃ of temperature with the aseptic seedling cultivation; Light application time 16h, per 35～40d subculture 1 time.
3. the dry ultralow temperature of the stripped stem apex embedding of horseradish test-tube plantlet according to claim 1 is preserved and regeneration and cultivation method; It is characterized in that: stem apex strips, and the stripped stem apex that under aseptic condition, cuts the about 1～3mm of diameter from the aseptic seedling of robust growth is a material.
4. the dry ultralow temperature of the stripped stem apex embedding of horseradish test-tube plantlet according to claim 1 is preserved and regeneration and cultivation method; It is characterized in that: cultivate in advance, the stem apex that strips is cultivated 1～2d with the MS solid culture medium that contains 0.3mol/L sucrose in advance under 20 ℃ or 25 ℃ of conditions.
5. the dry ultralow temperature of the stripped stem apex embedding of horseradish test-tube plantlet according to claim 1 is preserved and regeneration and cultivation method; It is characterized in that: calcium alginate embedding balling-up; Stripped stem apex after cultivating is in advance placed the no calcium MS culture fluid that contains 25g/L sodium alginate+2mol/L glycerine+0.8mol/L sucrose; Draw the no calcium MS that contains a stem apex with aseptic dropper or pipettor and cultivate drop to the culture fluid that contains 0.1mol/L calcium chloride+2mol/L glycerine+0.8mol/L sucrose; And keeping 25～30 min to make it into the embedding ball in 25 ℃, the about 4～5mm of each embedding ball size contains a stem apex.
6. the dry ultralow temperature of the stripped stem apex embedding of horseradish test-tube plantlet according to claim 1 is preserved and regeneration and cultivation method; It is characterized in that: the filtrated air fluidized drying; The embedding ball that will contain stem apex takes out, and is positioned on the aseptic filter paper on the sterile petri dish dry 3～5h in filtrated air stream.
7. the stripped stem apex embedding seasoning ultralow temperature of horseradish test-tube plantlet according to claim 1 is preserved and regeneration and cultivation method; It is characterized in that: the cryovial of packing into; After the filtrated air fluidized drying, with the embedding ball cryovial of packing into, each cryovial is adorned 10～20 embedding balls.
8. the dry ultralow temperature of the stripped stem apex embedding of horseradish test-tube plantlet according to claim 1 is preserved and regeneration and cultivation method; It is characterized in that: drop into nitrogen ultra low temperature and preserve, the cryovial that the embedding ball is housed is placed drop into liquid nitrogen behind freezing pipe support or the sandbag and carry out ultralow temperature and preserve.
9. the stripped stem apex embedding seasoning ultralow temperature of horseradish test-tube plantlet according to claim 1 is preserved and regeneration and cultivation method, and it is characterized in that: thaw, from liquid nitrogen, take out cryovial, 1.5～3min thaws in 38～40 ℃ of water-baths.
10. the dry ultralow temperature of the stripped stem apex embedding of horseradish test-tube plantlet according to claim 1 is preserved and regeneration and cultivation method; It is characterized in that: recover regenerating and culturing; Embedding ball after thawing is at MS+0.1mg/LBA+30g/L sucrose+6.5g/L agar of KNO3 950mg/L and NH4NO3 825mg/L; Recover in the medium of PH 5.8 to cultivate, after cultivating 8-10 days under the dark condition or the low light level, change over to again under the condition identical and cultivate earlier with successive transfer culture.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103081608A (en) * | 2013-02-26 | 2013-05-08 | 成都大学 | Germination processing method for horseradish seeds |
| CN103202225A (en) * | 2013-03-11 | 2013-07-17 | 上海市农业生物基因中心 | Ultralow-temperature storing, thawing and recultivating method for micro-stem tips of plants |
| CN111869660A (en) * | 2020-08-06 | 2020-11-03 | 浙江省亚热带作物研究所 | Method for embedding and low-temperature preservation of cymbidium tuberosum rhizome |
| CN111887154A (en) * | 2020-06-12 | 2020-11-06 | 江苏省中国科学院植物研究所 | Method for cryopreservation of embryonic callus of iris germanica |
| CN112655563A (en) * | 2021-01-14 | 2021-04-16 | 上饶师范学院 | Method for in vitro preservation of tetrastigma hemsleyanum test-tube plantlet |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101326906A (en) * | 2008-08-01 | 2008-12-24 | 集美大学 | Low temperature storage method of Porphyra haitanensis filament germplasm by encapsulation |
| CN102379281A (en) * | 2011-08-16 | 2012-03-21 | 上海市农业生物基因中心 | Ultralow-temperature preservation method and restoration culture method for carnation |
-
2012
- 2012-06-21 CN CN201210211725.6A patent/CN102696580B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101326906A (en) * | 2008-08-01 | 2008-12-24 | 集美大学 | Low temperature storage method of Porphyra haitanensis filament germplasm by encapsulation |
| CN102379281A (en) * | 2011-08-16 | 2012-03-21 | 上海市农业生物基因中心 | Ultralow-temperature preservation method and restoration culture method for carnation |
Non-Patent Citations (4)
| Title |
|---|
| C.SCOTTEZ.ET.AL: "Cryopreservation of Cold-Acclimated Shoot Tips of Pear in Vitro cultures after encapsulation-dehydration", 《CRYOBIOLOGY》, no. 29, 31 December 1992 (1992-12-31), pages 691 - 700 * |
| 王君晖等: "木本植物种质超低温保存的研究进展", 《世界林业研究》, no. 5, 31 December 1998 (1998-12-31), pages 6 - 10 * |
| 谷月等: "植物种质的包埋脱水超低温保存", 《植物生理学通讯》, vol. 43, no. 6, 31 December 2007 (2007-12-31), pages 1157 - 1160 * |
| 赵艳华等: "包埋干燥超低温保存苹果离体茎尖", 《园艺学报》, vol. 25, no. 1, 31 December 1998 (1998-12-31), pages 93 - 95 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103081608A (en) * | 2013-02-26 | 2013-05-08 | 成都大学 | Germination processing method for horseradish seeds |
| CN103081608B (en) * | 2013-02-26 | 2014-11-12 | 成都大学 | Germination processing method for horseradish seeds |
| CN103202225A (en) * | 2013-03-11 | 2013-07-17 | 上海市农业生物基因中心 | Ultralow-temperature storing, thawing and recultivating method for micro-stem tips of plants |
| CN111887154A (en) * | 2020-06-12 | 2020-11-06 | 江苏省中国科学院植物研究所 | Method for cryopreservation of embryonic callus of iris germanica |
| CN111869660A (en) * | 2020-08-06 | 2020-11-03 | 浙江省亚热带作物研究所 | Method for embedding and low-temperature preservation of cymbidium tuberosum rhizome |
| CN111869660B (en) * | 2020-08-06 | 2021-07-23 | 浙江省亚热带作物研究所 | Method for embedding and low-temperature preservation of cymbidium tuberosum rhizome |
| CN112655563A (en) * | 2021-01-14 | 2021-04-16 | 上饶师范学院 | Method for in vitro preservation of tetrastigma hemsleyanum test-tube plantlet |
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